Inclusion body formation of the recombinant protein expressed in pET28a in E.col

Hello...
I am new to the forum and really looking forward to get help through this..........I have expressed a protein of nearly 60kd in pet 28a in BL21(E.coli) but I am getting it as inclusion body. Please suggest me some way to solubilise the protein. I have tried expression at 16 with low IPTG..

Hi
If you have not yet got ur protein in soluble fraction, here is a suggestion. You can try cloning the gene in pET 32a/b/c. it has thioredoxin tag which helps to get the protein in soluble fraction. I have similar proble with my expressed protein. I have tried several methods but none of them worked. Only cloning in pET32 can help.
Otherwise you can try supplying sorbitol and betaine in the expression media. Some time it might help.

Maybe your T7 Promotor is to strong. The more is less and the less is more sometimes. And you can use a protein tag to make your ptotein soluble. Check for this pET-Manual. Use Glucose if your gene is toxic as an addition option.